Device for pathogen transport

ABSTRACT

This disclosure provides a method for transporting a pathogen under ambient conditions, by culturing the pathogen with an amoeba under conditions that favor the incorporation of the pathogen into a trophozoite, starving the amoeba until it encysts, then culturing under conditions that favor conversion of the amoeba back to a trophozoite. In one aspect, the conditions that favor incorporation of the pathogen into the cyst of the amoeba comprises contacting the pathogen with the amoeba in an iron rich environment. Virus and/or bacteria are pathogens that can be transported by the disclosed method. Amoeba that are useful in the disclosed methods include, without limitation  Acanthamoeba castellanii, Hartmannella vermiformis  and  Naegleria gruberi . The disclosed methods have utility in: transporting pathogens from military field hospitals and clinics to the laboratory; transporting pathogens from global satellite laboratories to clinical laboratories; long term storage of pathogens; enriching contaminated patient samples for pathogens of interest; biosurveillance and detection efforts.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/148,484, filed Jan. 6, 2014, the content of which is incorporatedherein by reference in its entirety.

STATEMENT OF FEDERAL SUPPORT

The United States Government has rights in the invention pursuant toContract No. DE-AC52-07NA27344 between the U.S. Department of Energy andLawrence Livermore National Security, LLC, for the operation of LawrenceLivermore National Laboratory.

BACKGROUND

New paradigms for early detection, prevention, and control at thehuman-environmental interface are needed to reduce global threats fromcurrent and emerging infectious diseases. For example, better storageand transport solutions are needed for pathogen-containing samplescollected from global satellite laboratories, health clinics andmilitary hospitals treating soldiers with persistent infections. Whileadequate collection and storage solutions are available for immediatetransport to the laboratory, these methods are extremely time sensitiveand often require refrigeration and/or cold storage.

Current methodologies for pathogen recovery and identification aredependent on obtaining sterile samples, which is often complicated inthe field by contamination of the material with patient or environmentalflora. In addition, existing methods are dependent on the use ofpathogen-specific transport media, which delays identification of newemerging biothreats in the field, where no such defined growth mediaexists.

Thus, a need exists for safe and convenient method for the transport ofpathogens at ambient temperatures. This disclosure satisfies this needand provides related advantages as well.

SUMMARY OF THE DISCLOSURE

Free-living amoebae are present worldwide, having been isolated fromsoil, fresh and salt water, and air. Amoebae are phagocytic andprimarily feed on bacteria, fungi and algae.

Some microorganisms have evolved to survive phagocytocis by amoebae andare known as Amoebae Resistant Microorganisms (ARM). In addition toLegionella and Mycobacterium spp., the list of ARM has recently beenshown to include Mimiviruses and Enteroviruses. The amoeba, Acanthamoebacastellanii, is known to serve as a reservoir for a number of pathogenicmicroorganisms in nature, and to play a role in their environmentalsurvival and dissemination. The ability of several human intracellularpathogens, including L. pneumophila and M avium, to infect and survivewithin A. castellanii has been well characterized. Amoebae have alsobeen shown to support the growth of many bacteria of interest tobiodefense (e.g. Francisella tularensis, Yersinia pestis, Coxiellabernetti) as well as a number of emerging pathogens (e.g. Klebsiellapneumoniae and Pseudomonas aeroginosa) in the laboratory. Importantly,environmental bio-surveillance programs increasingly identify amoebaecontaining Viable Nonculturable Organisms (VNCO), this suggests thatmany ARM remain to be identified and that amoebae growth may induce theintracellular bacteria to enter a metabolically dormant state.

Amoebae are known to exhibit a biphasic life style, existing astrophozoites in presence of abundant nutrients and as cysts in responseto desiccation and nutrient shortage. Multiple pathogens have been shownto persist in cysts for years and then emerge in response to favorableenvironmental conditions when the amoebae excyst. Amoebae cysts arecomposed of tough cellulose-like structures that are extremely resistantto biocide and mechanical lysis, which naturally protects any enclosedpathogens. In contrast to traditional transport media, where pathogensoften lose virulence factors present on extrachromosomal elements suchas plasmids or transposons, a number of studies have shown that growthin amoebae supports the virulence of ARM and their ability to invade andsurvive in host cells. This is believed to be due to similar selectivepressures present in amoebae and mammalian cells. It is currentlyaccepted that growth in amoebae provides an opportunity forintracellular pathogens to adapt to the intracellular niche enablingthem to subsequently resist killing by mammalian host cells. Developmentof amoeba cysts as a transport system has the advantages of using anenvironmentally tolerant system that supports pathogen growth andvirulence and is resistant to contamination with common skin flora.

Applicant provides a method for transporting a pathogen under ambientconditions, comprising, or alternatively consisting essentially of, oryet further consisting of, culturing the pathogen with an amoeba underconditions that favor the incorporation of the pathogen into atrophozoite of the amoeba and causing the trophozoite to encyst, andthen culturing under conditions that favor conversion of the amoeba cystto a trophozoite, which can release the engulfed pathogen. In oneaspect, the conditions that favor incorporation of the pathogen into thecyst of the amoeba comprises contacting the pathogen with the amoeba inan iron rich environment. Virus and/or bacteria are pathogens that canbe transported by the disclosed method. Amoeba that are useful in thedisclosed methods include, without limitation Acanthamoeba castellanii,Hartmannella vermiformis and Naegleria gruberi.

Also provided by this disclosure is a substantially homogenouspopulation or culture of amoeba trophozoites and/or cysts prepared bythe above method which comprises, or alternatively consists essentiallyof, or yet further consists of, an exogenous pathogen encysted in atrophozoite and/or an amoeba cyst. The composition comprising thesubstantially homogenous population or culture can further comprise, oralternatively consist essentially of, or yet further consist of acarrier, such as buffer, media or a device, as shown in FIG. 3. Thisdisclosure further provides a device as shown in FIG. 3 for culture andtransport of pathogens.

The disclosed methods have utility in: transporting pathogens frommilitary field hospitals and clinics to the laboratory; transportingpathogens from global satellite laboratories to clinical laboratories;long term storage of pathogens; enriching contaminated patient samplesfor pathogens of interest; bio-surveillance and detection efforts.

Further provided is a culture transport device comprising a housinghaving a first compartment and a second compartment, the first andsecond compartments being separated by a permeable membrane, wherein thesecond compartment comprises a removable cap comprising a samplingelongated member and a locking device, wherein when the locking deviceis activated, the permeable membrane is ruptured by the elongatedmember. In one aspect, the housing of the device is comprised of animpermeable temperature and pressure tolerant material such aspolypropylene.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates growth in B. mallei strains in amoeba trophozoites.Time points were taken daily and reflect the means and standarddeviations of triplicate assays. All experiments were conducted at least3 times. % Survival=cfu at Tx/cfu at TOX100.

FIG. 2 illustrates survival of a cyclically passaged population of B.pseudomallei in human primary monocytes. The population was grown inamoeba cysts for 21 days, harvested then grown in lab media for 24 hoursbefore being used to infect fresh amoeba again. At each passage analiquot of the passaged population was used to infect human monocytes inparallel to lab-grown bacteria and freshly harvested amoeba-grownbacteria. Histograms and error bars represent the means and standarddeviations of assays done in triplicate. Asterisks indicate the onlystatistically significant differences between samples.

FIG. 3 depicts a device of this disclosure that can be utilized for thesafe culturing and transport of the amoeba cysts.

DETAILED DESCRIPTION Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook, Fritsch andManiatis (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) edition;F. M. Ausubel, et al. eds. (1987) Current Protocols In MolecularBiology; the series Methods in Enzymology (Academic Press, Inc.): PCR 2:A Practical Approach (1995) (M. J. MacPherson, B. D. Hames and G. R.Taylor eds.); Harlow and Lane, eds. (1988) Antibodies, A LaboratoryManual; Harlow and Lane, eds. (1999) Using Antibodies, a LaboratoryManual; and R. I. Freshney, ed. (1987) Animal Cell Culture.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate. It is tobe understood, although not always explicitly stated that all numericaldesignations are preceded by the term “about”. It also is to beunderstood, although not always explicitly stated, that the reagentsdescribed herein are merely exemplary and that equivalents of such areknown in the art.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the likeinclude the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above.

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination when used for the intendedpurpose. Thus, a composition consisting essentially of the elements asdefined herein would not exclude trace contaminants or inert carriers.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “isolated” means separated from constituents, cellular andotherwise, in which the cell, tissue, polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, which arenormally associated in nature. For example, an isolated polynucleotideis separated from the 3′ and 5′ contiguous nucleotides with which it isnormally associated in its native or natural environment, e.g., on thechromosome. As is apparent to those of skill in the art, a non-naturallyoccurring polynucleotide, peptide, polypeptide, protein, antibody orfragment(s) thereof, does not require “isolation” to distinguish it fromits naturally occurring counterpart. An isolated cell is a cell that isseparated form tissue or cells of dissimilar phenotype or genotype.

The term “propagate” means to grow or alter the phenotype of a cell orpopulation of cells. The term “growing” refers to the proliferation ofcells in the presence of supporting media, nutrients, growth factors,support cells, or any chemical or biological compound necessary forobtaining the desired number of cells or cell type. In one embodiment,the growing of cells results in the regeneration of tissue.

The terms “culturing” or “incubating” refer to the in vitro propagationof cells or organisms on or in media of various kinds. It is understoodthat the descendants of a cell grown in culture may not be completelyidentical (i.e., morphologically, genetically, or phenotypically) to theparent cell. By “expanded” is meant any proliferation or division ofcells.

A “composition” is also intended to encompass a combination of acompound or composition and another carrier, e.g., a solid support suchas a culture plate or biocompatible scaffold, inert (for example, aculture plate) or active, such as an adjuvant, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvantor the like. Carriers also include pharmaceutical excipients andadditives proteins, peptides, amino acids, lipids, and carbohydrates(e.g., sugars, including monosaccharides, di-, tri-, tetra-, andoligosaccharides; derivatized sugars such as alditols, aldonic acids,esterified sugars and the like; and polysaccharides or sugar polymers),which can be present singly or in combination, comprising alone or incombination 1-99.99% by weight or volume. Exemplary protein excipientsinclude serum albumin such as human serum albumin (HSA), recombinanthuman albumin (rHA), gelatin, casein, and the like. Representative aminoacid/antibody components, which can also function in a bufferingcapacity, include alanine, glycine, arginine, betaine, histidine,glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine,valine, methionine, phenylalanine, aspartame, and the like. Carbohydrateexcipients are also intended within the scope of this invention,examples of which include but are not limited to monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) andmyoinositol.

“Substantially homogeneous” describes a population of amoeba in whichmore than about 50%, or alternatively more than about 60%, oralternatively more than 70%, or alternatively more than 75%, oralternatively more than 80%, or alternatively more than 85%, oralternatively more than 90%, or alternatively, more than 95%, oralternatively more than 97% of the amoeba are of the same or similarphenotype.

The term “effective amount” refers to a concentration or amount of areagent or composition, such as a composition as described herein, cellpopulation or other agent, that is effective for producing an intendedresult, including cell growth and/or differentiation in vitro or invivo, or for the treatment of a disease or condition as describedherein. It will be appreciated that the number of cells to beadministered will vary depending on the specifics of the disorder to betreated, including but not limited to size or total volume/surface areato be treated, as well as proximity of the site of administration to thelocation of the region to be treated, among other factors familiar tothe medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orcomposition to achieve its intended result, e.g., the differentiation ofcells to a pre-determined cell type.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein. Other animalsinclude, simians, bovines, ovines, equines, canines, felines, andmurines.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms that are not only compatible with the cells andother agents to be administered therapeutically, but also are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable carrierssuitable for use in the present invention include liquids, semi-solid(e.g., gels) and solid materials (e.g., cell scaffolds and matrices,tubes sheets and other such materials as known in the art and describedin greater detail herein). These semi-solid and solid materials may bedesigned to resist degradation within the body (non-biodegradable) orthey may be designed to degrade within the body (biodegradable,bioerodable). A biodegradable material may further be bioresorbable orbioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids(water-soluble implants are one example), or degraded and ultimatelyeliminated from the body, either by conversion into other materials orbreakdown and elimination through natural pathways.

Methods

For the purpose of illustration only, A. castellanii, the select agentpathogen Burkholderia pseudomallei, and the emerging pathogenAcinetobacter baumannii, A. castellanii cysts can act as a naturaltransport system for pathogens from field locations to the laboratory atroom temperature, e.g., from about 23° C. to about 45° C., oralternatively from about 23° C. to about 42° C., or about 37° C. Thesepathogens survive long-term in amoebae cysts and can be recovered postexcystment for testing or other manipulation as desired. As shown inmore detail below, Applicant has used the disclosed method to recoverbacteria and confirmed phenotype to ensure no loss of viability orvirulence. Non-limiting examples of bacteria for transport using theabove method include, without limitation A. baumannii (Bouvet andGrimont ATCC 9955) and B. pseudomallei, (ATCC 23343).

Thus, in one aspect, Applicant provides a method for transporting apathogen under ambient conditions e.g., from about 23° C. to about 45°C., or alternatively from about 23° C. to about 42° C., or about 37° C.,comprising, or alternatively consisting essentially of, or yet furtherconsisting of, culturing the pathogen with an amoeba under conditionsthat favor the incorporation of the pathogen into a trophozoite of theamoeba, causing the trophozoite to encyst, and then culturing underconditions that favor the conversion of the amoeba cyst to atrophozoite. As is apparent to those of skill in the art, a trophoziteis the active feeding and motile stage of the ameboid while the cyststage is the dormant, non-feeding or mobile stage in the life cycle.

Applicant has discovered that the conditions that favor incorporation ofthe pathogen into the trophozoite comprise or are enhanced by, oralternatively consist essentially of, or yet further consist of,contacting the pathogen with the amoeba in an iron rich environment. Asused herein, the term “iron rich” intends an environment having, ascompared to conventional media for the growth and/or maintenance ofamoeba, at least 2×, or alternatively at 3×, or alternatively at least4×, or alternatively at least 5×, or alternatively at least 6×, oralternatively at least 7×, or alternatively at least 8×, oralternatively at least 9×, or alternatively at least 10×, concentrationof iron in the media, or alternatively from about 2× to 10×, oralternatively at least about 3× to 10×, or alternatively at least about4× to 10×, or alternatively at least about 5× to 10×, or alternativelyat least about 6 to 10× of an iron source in the media. Alternatively,the iron is present in an amount of at least about 0.005 M, or at leastabout 0.01M, or alternatively at least about 0.02M, or alternatively atleast about 0.03M, or alternatively at least about 0.04M, oralternatively at least about 0.05M, or alternatively at least about0.06M, or alternatively at least about 0.07M, or alternatively fromabout 0.01M to 0.07M, or alternatively from about 0.02M to about 0.06M,or alternatively from about 003M to about 0.06M, or alternatively about0.05 M iron, e.g., 0.05M ferrous ammonium sulfate (FeAmSO₄).

Applicant has discovered that when the amoeba and the pathogen arecultured in conditions of iron rich environment, the amoeba quicklyengulfs the pathogen. The culturing is accomplished under ambientconditions, depending on the environment of the pathogen. For example,the amoeba can be transported from a tropical location as a cyst andthen converted to the active growing form (trophozoite) by addingnutrient rich media. Thus, the pathogen can be added to the amoeba underconditions that favor the growth and replication of the amoeba and theculture media is changed to nutrient rich and an iron source is added tothe culture to provide the iron rich environment. When the nutrients inthe culture media have been depleted, the amoeba will convert into acyst, which is environmentally tolerant and can be easily transported.In a further aspect, antimicrobial or antibiotics can be added to removeall active pathogen in the culture just prior to or after cystformation. As noted before, the trophozoite containing the pathogen willthen encyst and can then be transported under ambient conditions. Thecyst is converted back to the actively growing trophozoite form and thepathogen is released from the trophozoite by culturing the cysts underin iron and nutrient rich media. In one aspect, a solution to lyse thetrophozoite, such as a 0.5% saponin solution can be used to release thepathogen from the trophozoite.

For the purpose of illustration only, Applicant has found that byculturing at least 0.5×10⁶ or alternatively at least 1.0×10⁶ pathogensper ml iron-rich media, the amoeba and pathogen will replicate and theamoeba will engulf the pathogen and convert to a cyst in several hours.After the cyst is converted into an amoeba trophozoite, the pathogen arereleased into the culture media where they can be isolated andcharacterized using conventional microbial techniques.

The above methods are useful to transport virus or bacteria, e.g.,Legionella, Mycobacterium, Francisella, Yersinia, Coxiella, Klebsiella,Pseudomonas, Burkeholderia and Acinetobacer. In one aspect, the amoebais selected from the group consisting of Acanthamoeba castellanii,Hartmannella vermiformis and Naegleria gruberi. For added convenience,the entire process and transport can be accomplished in conventionalmodified cryopreservation vials (see FIG. 3).

This disclosure also provides a device or container for the safetransport and storage of pathogens or microbes using the methods asdisclosed herein. As shown in FIG. 3, the device is a multi-compartmentcontainer containing at least 2 compartments having at least oneremovable cap or tip. The shape of the device is not critical to thedesign. For example, the container can be cylindrical. The outer wallsof the container (housing) are made of an impermeable material that isresistant to temperature and pressure fluctuations. Examples ofmaterials include without limitation polypropylene or other plastic. Theat least two-compartment container is separated by a liquid impermeablemembrane or barrier that is permeable to pressure. One cap that isremovable has contained in it an elongated swabbing device, such as acotton swab, for sampling the pathogen or microbe. It is fitted withlocking device such as a luer lock that when closed in the lockedposition, will push the swab containing the pathogen into the othercompartment through the permeable membrane, e.g., a thin rubber, plasticor the like. At least one other compartment is configured to contain theamoeba cysts in a buffer solution and while the other is configured tocontain the iron and nutrient rich medium. When needed, the cap isremoved and one compartment is opened. The swab is used to collect thesample. The swab is returned to the inside of the container and it islocked using the locking device such as the luer lock, or any other twostep lock, thereby forcing the swab through the membrane and allowingthe nutrient rich medium to fall into the other compartment. Mixing thecontainer will bring the amoeba cysts in the bottom compartment tocontact the iron-rich medium and the sample resulting in amoebaexcystment and microbial phagocytosis by amoeba trophozoites.

Using FIG. 3 as example, shown therein is a two compartment cylindricalcontainer having a compartment (101) that contains the amoeba in buffer(102). An additional compartment (104) has at one end a removable cap(107) with a sampling device or swab (106) and iron rich media (108).The cap (107) contains a locking device (105). Separating thecompartment is permeable membrane (103) that is punctured when thelocking device (105) of the cap is activated allowing the iron richmedia to flow from one compartment (104) into the other (101) and thepathogen and the amoeba to come in contact with each other.

Materials and Methods Media

Rich Media: Modified Peptone-Yeast-Glucose (PYG)

Protease peptone 20 g Yeast Extract 1 g Add 900 ml of dd H₂O. Autoclaveand cool down to at least 55° C. Add the following: Sodium citrate 1 g0.4M Magnesium Sulfate (MgSO₄) 10 ml 0.05M Calcium chloride (CaCl₂) 8 ml0.05M Ferrous Ammonium Sulfate (FeAmSO₄) 10 ml (Filter sterilize, do notautoclave) 0.25M Dibasic Sodium phosphate ((Na)₂HPO₄) 10 ml 0.25MMonobasic Potassium phosphate (KH₂PO₄) 10 mlAdjust pH to 6.5 exactly. Add 50 ml of 2 M glucose. Filter sterilizethrough a 0.22 μm filter.

High Salt Buffer

Mix the following:

Sodium citrate 1 g 0.4M Magnesium Sulfate (MgSO₄) 10 ml 0.05M Calciumchloride (CaCl₂) 8 ml 0.05M Ferrous Ammonium Sulfate (FeAmSO₄) 10 ml(Filter sterilize, do not autoclave) 0.25M Dibasic Sodium phosphate((Na)₂HPO₄) 10 ml 0.25M Monobasic Potassium phosphate (KH₂PO₄) 10 mlAdd 950 ml of dd H₂O. Adjust pH to 6.5 exactly. Filter sterilize througha 0.22 μm filter.

The ability of B. pseudomallei (Bp) and A. baumannii (Ab) isolates tosurvive in A. castellanii (AC) trophozoites and cysts can be confirmedby conducting infection and intracellular survival assays. For example,amoeba trophozoites are grown in Peptone-Yeast-Glucose medium at roomtemp (RT) in the dark. Amoeba are then seeded at a concentration ofapproximately 10⁵ per ml per well in 24-well plates in PYG broth at roomtemperature.

Bacteria are grown overnight (o/n) in Nutrient broth (NB for both Ab andBp) available from Difco-Invitrogen) broth at 37° C. with shaking.

The PYG medium can be removed by aspiration from the Ac plates andreplaced by 1 ml of High Salt Buffer (HSB) per well and the platesincubated at 37° C. for 1 hr. 100 μl of bacterial cultures are added peramoeba well to achieve a multiplicity of infection (MOI) of 10 and thenincubated for 30 min at 37° C. to allow bacterial internalization thenwashed 1× with HSB. Media is replaced by fresh HSB containing 100 μg/mlgentamicin and the plates incubated for 2 hours to kill extracellularbacteria. Chloroamphenicol can be used for gentamicin resistant strains.

The amoeba will is washed 1× with HSB then lysed with 0.5% Saponinrelease intracellular bacteria. Conventional microbiological techniques,such as plating on nutrient agar plates can be used to determinebacterial colony forming units (CFU).

To determine intracellular survival, 1 ml of fresh HSB can be added toparallel wells instead of immediate lysis at time point zero. Wells arethen incubated for varying time points prior to lysis and platting.

% bacterial survival=CFU at 24 hr/CFU at time zero

Amoeba trophozoites typically encyst within 2 days after nutrientdepletion. To confirm the ability of the bacteria to survive long termin amoeba cysts, plates can be incubated at temperatures ranging from 4°C. to 42° C. to demonstrate environmental tolerance. One set of wellscan be lysed at weekly time points. At each time point, amoeba cystswill be centrifuged for 5 min at 1000 g. The medium will be decanted andreplaced with PYG to allow excystment. Amoeba cysts are incubated at 37°C. until the first sign of turbidity or for 48 hours to allow the amoebato excyst and the bacteria to be released into the medium. Intracellularbacteria can be recovered by platting dilutions on nutrient agar.

Genetic Stability and Storage

To confirm the suitability of A. castellanii cysts to function as atransport system for pathogens, four isolates of Burkholderiapseudomallei and one isolate of Acinetobacter baumanii were sequencedbefore and after they were grown in amoebae for one month, and thegenomic sequences compared to identify any genetic differences. TheIllumina Hi-Seq 2000 platform was used for sequencing and at least 100×genome coverage was targeted to ensure high confidence mutation analysisin the isolates. A preliminary analysis of the A. baumanii isolates andone pair of the B. pseudomallei isolates follows.

A. baumanii

The Burrows-Wheeler Aligner (BWA) program was used to align the sequencereads from both isolates against the Reference Sequence genome for A.baumannii strain ACICU. A custom code was developed to count thenucleotides of each type that aligned to each reference genome position,plus the numbers of deletions (gaps) at each position. This produced atable of allele frequencies for each isolate at each position. The codethen looked for positions where the major allele differed between theisolates. Out of 3.9 MB in the reference genome, about 29,000 suchpositions were identified.

Positions that had 5 or fewer reads were filtered out from eitherisolate mapped to them, since allele frequency estimates based on smallread counts are inherently unreliable. This excluded all but the 331locations. For each location, a chi-squared statistic and associatedP-value were computed, estimating the probability that the observeddifference in observed allele frequencies between the un-passaged andamoeba-passaged isolates could have occurred by chance.

Of immediate note is that, for almost all positions selected forcandidate mutations, the major allele frequency ranges from 45% to 65%;there is only one site (1891835) where 100% of the reads have one allelein the un-passaged isolate and a different allele for most of the readsin the passaged isolate. Sorting the table in descending order of majorallele frequency for the un-passaged isolate, reveals that the 3positions with the highest major allele frequency (MAF) all haverelatively small numbers of reads mapped to them. Any evidence foractual mutations at these sites is pretty weak.

In addition, when re-sorting the table by genome position, Applicantnoted that the locations of the candidate mutations were clustered intosmall genome regions. The distribution of these regions suggested thatthey map to insertion (IS) elements, or other repeated sequences. Onexamining the GenBank annotations for the A. baumannii ACICU genome,positions 262878-264180 was annotated as a hypothetical protein.However, when performing BLAST queries of this protein sequence againstGenBank data, it was found that analogous proteins are annotated asantibiotic resistance islands, genomic resistance islands andtransposons. These data suggest that A. baumanii reads mapping to thisregion are hitting a transposon with an imperfect copy elsewhere in thegenome, i.e., BWA could be mapping them arbitrarily to either copy,giving the appearance of a mixed population.

Another region, 1109338-1165207, is somewhat larger (56 kB), and maps tomultiple genes, so it's less obvious that there it is a repeat sequence.However, generation of a dotplot from performing BLAST queries of theACICU genome against itself indicates a string of duplicated sequences;one set of copies in the region 1150000-1155000, the other from2900000-2935000 (which includes the other large stretch of candidatemutations). These data suggest a similar pattern, where reads are mappedarbitrarily to the different copies and thus appear to indicatemutations, when actually they're just reflecting the variation betweenparalogs.

In summary, our initial analyses suggest that all the candidatemutations observed are resulting either from either noise variation whenthe number of mapped reads is small, or from variation between paralogswithin the reference genome. So at least for this pair of isolates,there is no evidence for mutations resulting from growth in amoebae.

B. pseudomallei

Similar to the approach used for A. baumannii isolates, BWA was used toalign the sequence reads from the two isolates of B. pseudomallei strainPHLS17—one passaged in amoeba and the other grown in standard labbroth—against the Reference Sequence genome for B. pseudomallei strainK96243. A custom program was created to tabulate, at each position inthe two K96243 chromosomes, the allele frequencies for each nucleotideand the frequency of deletions (gaps). About 29,000 positions (out of7.1 million) for which the major allele differed between the two sets ofreads. However, these differences are unlikely to represent actualmutations. Since B. pseudomallei are characterized by extensiverecombination and duplication of genome regions, many sequence reads(about 35,000 in this case) align to multiple locations in the genome.Subsequent mutations in the duplicated regions in distant ancestors ofthe current PHLS17 strain cause slight variations between the multiplegenome sequences aligned to a sequence read. Similar to our observationson the analysis of the A. baumannii sequences, these variations betweenparalogous regions can masquerade as mutations between isolates. B.pseudomallei genomes have vastly more internal duplication than A.baumannii genomes, so the number of potential false mutations is muchlarger.

Long Term Stability

F. tularensis

In contrast to B. pseudomallei and A. baumanii, F. tularensis is a veryfastidious organism that is often hard to grow in the laboratory. TheSchu S4 strain, which is the typical type A strain used in research, wasused as well as three clinical strains isolated from human Tularemiaoutbreaks in Utah and minimally manipulated in the laboratory. One ofthese strains (80700069) has been identified as type A2, while the restare all type A1 strains. Previous work has shown that all these strainsare capable of entry and replication within amoeba (El-Etr, S. H. et.al. (2009) Appl Environ Microbiol. 75:7488-7500). In addition, with theexception of strain 80700069, all the strains cause the rapid encystmentof A. castellanii.

All clinical strains are shown to survive intracellularly in amoebacysts up to six weeks post infection (Table 1) at temperatures rangingfrom 26 to 42° C. The Schu S4 strain was not recovered from amoeba cystsafter three weeks post infection. This observation may be explained bythe fact that the Schu S4 strain has been propagated under laboratoryconditions for almost 70 years since its initial isolation from aclinical case and that some loss of virulence is to be expected.

TABLE 1 Recovery of F. tularensis type A strains from amoeba cysts atweekly intervals after initial infection. NG denotes no growth inexperimental wells after addition of rich medium, plus signs indicatesuccessful bacterial recovery and Al or A2 indicates strain type. StrainWeek 1 Week 2 Week 3 Week 4 Week 5 Week 6 SchuS4 (A1) + + + NC, NC, NG70102163(A1) + + + + + + 80700069 (A2) + + + + + + 80502541(Al) + + + + + +B. mallei

Though not as fastidious as F. tularensis, B. mallei strains are not asenvironmentally tolerant as B. pseudomallei and for a long time theorganisms were thought to have a strict requirement for a mammalianhost. Recent data from Australia however indicate the organism maysurvive long enough in the environment to infect incidental hosts.

Two strains of B. mallei from the ATCC were used to investigate theability to survive and replicate in amoeba (ATCC (15310 and 10399) andtwo clinical strains isolated from India and Turkey. Of these strains,only the Turkish strain (NCTC 10260) is known to be isolated from aninfected human, the Indian strain having been isolated from a mule. Therest of the strains have been isolated from horses, the natural hosts ofB. mallei.

The data indicate that all the B. mallei strains we tested can surviveand replicate well in amoeba which has never been reported before.

Long-term growth experiments examining the ability of B. mallei strainsto survive in amoeba cysts indicate that all the strains tested are ableto survive for 3 weeks post infection at temperatures ranging from 26 to42° C. (Table 2). Although clinical strains were recovered up to sixweeks post infection, recovery of the ATCC strains after three weeks wasinconsistent, especially at 42° C. which may again be explained by lossof virulence since both strains were originally isolated more than 50years ago.

TABLE 2 Recovery of B. mallei strains from amoeba cysts at weeklyintervals after initial infection NG denotes no growth in experimentalwells after addition of rich medium, plus signs indicate successfulbacterial recovery. Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 ATCC15310 + + + NG NG NG ATCC 10399 + + + NG NG NG NCTC 10260 + + + + + +CDC 85 + + + + + +

On a final note, even though both F. tularensis and B. mallei clinicalstrains can survive in amoeba cysts up to six weeks, recovery of thebacterial isolates after transport has yet to be done at 37° C. in thelaboratory. Attempts to recover both pathogens at higher temperatureswere not successful, possibly due to the sensitivity of both organismsto high temperatures when they are not protected inside amoebae cysts.

Sampling and Transport of Skin Flora

Experiments were performed with A. baumanii and B. pseudomallei inpresence of human skin flora. Sterile cotton swabs were wetted with HSBand used to collect skin flora from the forearms of healthy adultvolunteers. The collected bacteria were used to inoculate 1 ml of HSBand the samples were randomized then numerically labeled per IRBrequirements. An aliquot of the buffer was then plated directly onnutrient agar (to determine number of bacteria added) and the restinoculated with dilutions of either A. baumanii or B. pseudomalleicultures and added to 10⁶ amoeba trophozoites previously seededovernight. The amoebae were then incubated for 30 days at temperaturesranging from 26 to 42° C. The dilutions of A. baumanii or B.pseudomallei cultures were also plated to determine the bacterialnumbers added. After one month, rich media was added to all wells. Uponobserving signs of turbidity, dilutions of the supernatants were platedto assess colony-forming units (cfu) as well as streaked for isolationto enable gram staining and bacterial identification.

Results indicate that in each experiment, Applicant was able to detectas little as 430 cfu A. baumanii (Table 3) or 177 cfu of B. pseudomallei(Table 4). This makes the multiplicity of infection <0.001 (MOI numberof bacteria/number of amoeba). These results were consistent attemperatures ranging from 26 to 42° C. In addition, in almost all casesthe amoebae were able to consume all the skin bacteria while preservingthe pathogen. With the exception of one case (Table 4, Subject 9)negligent amounts of flora were recovered. The amoeba was therefore ableto purify the sample from skin flora and enrich for the pathogen ofinterest. Interestingly, analysis of the colonies recovered from subject9, showed that more than 95% of the skin colonies recovered wereStaphylococcus aureus indicating the subject was a carrier therebygiving us positive preliminary data about the ability of gram-positivepathogens to survive in amoebae cysts.

TABLE 3 A. baumanii and bacterial skin flora recovered after incubationin amoebae cysts for one month. “Added” represents the cfu of A.baumanii or skin flora inoculated into amoebae cysts. “Recovered”represents the cfu recovered after a 30-day incubation. TNTC: cfu toonumerous to count >1000 cfu. A. baumanii Subject 1 Subject 2 Subject 3Subject 4 Subject 5 Subject 6 added/ added/ added/ added/ added/ added/added/ recovered recovered recovered recovered recovered recoveredrecovered 4.3e⁷/TNTC 268/0 48/0 27/2 89/0 64/1 37/0 4.3e⁶/TNTC 268/148/0 27/0 89/1 64/0 37/0 4.36⁵/TNTC 268/3 48/1 27/0 89/0 64/2 37/14.3e⁴/TNTC 268/0 48/2 27/0 89/0 64/0 37/0 4.36³/TNTC 268/0 48/0 27/089/2 64/0 37/0 4.3e²/TNTC 268/0 48/0 27/0 89/0 64/0 37/0

TABLE 4 B. pseudomallei and bacterial skin flora recovered afterincubation in amoebae cysts for one month. “Added” represents the cfu ofB. pseudomallei or skin flora inoculated into amoebae cysts. “Recovered”represents the cfu recovered after a 30-day incubation. TNTC: cfu toonumerous to count >1000 cfu. A. baumanii Subject 1 Subject 2 Subject 3Subject 4 Subject 5 Subject 6 added/ added/ added/ added/ added/ added/added/ recovered recovered recovered recovered recovered recoveredrecovered 4.3e⁷/TNTC 268/0 48/0 27/2 89/0 64/1 37/0 4.3e⁶/TNTC 268/148/0 27/0 89/1 64/0 37/0 4.3e⁵/TNTC 268/3 48/1 27/0 89/0 64/2 37/14.3e⁴/TNTC 268/0 48/2 27/0 89/0 64/0 37/0 4.3e³/TNTC 268/0 48/0 27/089/2 64/0 37/0 4.3e²/TNTC 268/0 48/0 27/0 89/0 64/0 37/0 B. pseudomalleiSubject 7 Subject 8 Subject 9 Subject 10 Subject 11 Subject 12 added/added/ added/ added/ added/ added/ added/ recovered recovered recoveredrecovered recovered recovered recovered 1.77e⁷/TNTC 115/0 87/1 287/13109/2 16/0 71/0 1.77e⁶/TNTC 115/0 87/0 287/37 109/1 16/0 71/01.77e⁵/TNTC 115/2 87/0 287/28 109/0 16/0 71/0 1.77e⁴/TNTC 115/1 87/1287/32 109/2 16/1 71/1 1.77e³/TNTC 115/0 87/0 287/43 109/0 16/0 71/01.77e²/TNTC 115/0 87/0 287/38 109/0 16/0 71/0

Changes in Antimicrobial, Biocide Resistance and Macrophage Survival toEnsure Lack of Phenotypic and Virulence Changes During Amoebae Growth.

Applicant has shown that survival in amoebae cysts does not increase theresistance Acinetobacter baumannii and Burkholderia pseudomallei tobiocides and antimicrobials. To determine whether the enhancement in theability of B. pseudomallei to survive in macrophages is a permanentchange, amoeba-grown bacteria was inoculated into nutrient broth andgrown under standard laboratory conditions for 24 hr. The bacteria werethen used to re-infect fresh amoeba trophozoites. This cycle wasrepeated four times and at the end of each cycle the passaged B.pseudomallei population was tested for macrophage survival in parallelto regular lab-grown bacteria and bacteria freshly harvested fromamoeba. The results show that after each passage in the lab media thesurvival of amoeba-grown bacteria returned to the levels of the brothgrown strain within 24 hours (FIG. 2). These data suggest that theobserved survival is due to gene regulation rather that to permanentmutations.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification, improvement and variation of the inventions embodiedtherein herein disclosed may be resorted to by those skilled in the art,and that such modifications, improvements and variations are consideredto be within the scope of this. The materials, methods, and examplesprovided here are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

All publications, patent applications, patents, and other referencesmentioned herein are expressly incorporated by reference in theirentirety, to the same extent as if each were incorporated by referenceindividually. In case of conflict, the present specification, includingdefinitions, will control.

REFERENCES

-   1. Rodriguez-Zaragoza S (1994) Ecology of free-living amoebae. Crit    Rev Microbiol 20: 225-241.-   2. Greub G, Raoult D (2004) Microorganisms resistant to free-living    amoebae. Clin Microbiol Rev 17: 413-433.-   3. Cirillo J D, Falkow S, Tompkins L S (1994) Growth of Legionella    pneumophila in Acanthamoeba castellanii enhances invasion. Infect    Immun 62: 3254-3261.-   4. Goy G, Thomas V, Rimann K, Jaton K, Prod′hom G, et al. (2007) The    Neff strain of Acanthamoeba castellanii, a tool for testing the    virulence of Mycobacterium kansasii. Res Microbiol 158: 393-397.-   5. El-Etr S H, Margolis J J, Monack D, Robison R A, Cohen M, et    al. (2009) Francisella tularensis type A strains cause the rapid    encystment of Acanthamoeba castellanii and survive in amoebal cysts    for three weeks postinfection. Appl Environ Microbiol 75: 7488-7500.

What is claimed is:
 1. A culture transport device comprising housing having a first compartment and a second compartment, the first and second compartments being separated by a permeable membrane, wherein the second compartment comprises a removable cap comprising a sampling elongated member and a locking device, wherein when the locking device is activated, the permeable membrane is ruptured by the elongated member.
 2. The device of claim 1, wherein the housing of the device is comprised of an impermeable material.
 3. The device of claim 1, wherein the impermeable material is polyethylene.
 4. A method for transporting a pathogen, comprising contacting the pathogen with the elongated swabbing member of the device of any one of claims 1-3, and inserting the swab into the second compartment and closing the cap and engaging the locking member. 